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81.
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We have examined several mutants in the switch I, switch II region of rat kinesin. Pre-steady-state kinetic analysis of association and dissociation of an N256K mutant with nucleotides and microtubules demonstrates that the mutation blocks microtubule stimulation of nucleotide release and ATP hydrolysis without affecting other kinetic parameters. The results suggest that ADP release on one head may be coupled to structural changes on the other head to stimulate ATP hydrolysis. Mutations at Glu(237), a residue predicted to participate in a hydrogen-bond interaction critical for nucleotide processing, reduced or abolished microtubule-dependent ATPase activity with only minor effects on pre-steady-state rates of nucleotide release or binding. Mutations at Glu(200), a residue that could serve as an alternate electron acceptor in the above-mentioned hydrogen-bond interaction, had small effects on microtubule-dependent ATPase activity despite modestly reducing the rate at which microtubule-stimulated nucleotide release occurs. These results further clarify the pathway of coupling of ATP hydrolysis to force production. 相似文献
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Cheng L Naumann TA Horswill AR Hong SJ Venters BJ Tomsho JW Benkovic SJ Keiler KC 《Protein science : a publication of the Protein Society》2007,16(8):1535-1542
A method to rapidly screen libraries of cyclic peptides in vivo for molecules with biological activity has been developed and used to isolate cyclic peptide inhibitors of the ClpXP protease. Fluorescence activated cell sorting was used in conjunction with a fluorescent reporter to isolate cyclic peptides that inhibit the proteolysis of tmRNA-tagged proteins in Escherichia coli. Inhibitors shared little sequence similarity and interfered with unexpected steps in the ClpXP mechanism in vitro. One cyclic peptide, IXP1, inhibited the degradation of unrelated ClpXP substrates and has bactericidal activity when added to growing cultures of Caulobacter crescentus, a model organism that requires ClpXP activity for viability. The screen used here could be adapted to identify cyclic peptide inhibitors of any enzyme that can be expressed in E. coli in conjunction with a fluorescent reporter. 相似文献
86.
Zabotina OA van de Ven WT Freshour G Drakakaki G Cavalier D Mouille G Hahn MG Keegstra K Raikhel NV 《The Plant journal : for cell and molecular biology》2008,56(1):101-115
The function of a putative xyloglucan xylosyltransferase from Arabidopsis thaliana (At1g74380; XXT5) was studied. The XXT5 gene is expressed in all plant tissues, with higher levels of expression in roots, stems and cauline leaves. A T-DNA insertion in the XXT5 gene generates a readily visible root hair phenotype (root hairs are shorter and form bubble-like extrusions at the tip), and also causes the alteration of the main root cellular morphology. Biochemical characterization of cell wall polysaccharides isolated from xxt5 mutant seedlings demonstrated decreased xyloglucan quantity and reduced glucan backbone substitution with xylosyl residues. Immunohistochemical analyses of xxt5 plants revealed a selective decrease in some xyloglucan epitopes, whereas the distribution patterns of epitopes characteristic for other cell wall polysaccharides remained undisturbed. Transformation of xxt5 plants with a 35S::HA-XXT5 construct resulted in complementation of the morphological, biochemical and immunological phenotypes, restoring xyloglucan content and composition to wild-type levels. These data provide evidence that XXT5 is a xyloglucan alpha-1,6-xylosyltransferase, and functions in the biosynthesis of xyloglucan. 相似文献
87.
Kenneth W. Gasser Leonard B. Kirschner 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1987,157(4):469-475
Summary The relationship between alkaline phosphatase and environmental salinity was examined in the rainbow trout and the migratory rainbow (steelhead),Salmo gairdneri. The enzyme activity in tissues involved in osmoregulation was strongly correlated with the adaptation salinity and thus to the degree of salt and fluid transport in those tissues. After transfer from freshwater to seawater, the specific activity of the enzyme increased over 260% in the intestine, decreased by 50% in kidney, and was unchanged in the liver, an organ not directly involved in osmoregulation. The sea-run steelhead trout response was similar to the nonmigratory rainbow; although, the pre-migratory transformation (smoltification) had no effect on enzyme activity. Amino acid inhibitors of alkaline phosphatase significantly reduced fluid absorption in the isolated intestine of rainbow trout, reaffirming the relationship between the enzyme and fluid movement. Electrophoretic identification of trout alkaline phosphatase isozymes, clearly distinguishes the enzyme from different tissue origins. However, from the analysis of intestinal electrophoretic patterns, osmoregulatory adjustments are not associated with the induction of new alkaline phosphatase isozymes, or in the large scale preferential stimulation of one of the two existing intestinal isozymes over the other. 相似文献
88.
Biological invasions can alter ecosystem functions such as litter decomposition and nutrient cycling, but little is known about how invader abundance influences the impact on the ecosystem. It is often assumed that impacts are proportional to invasion density, but this assumption has never been tested and has little justification. We tested the hypothesis that the microbial community structure and function of a mixed hardwood forest soil changed after invasion by Japanese barberry (Berberis thunbergii), an invasive shrub commonly found in eastern hardwood forests, and that changes were proportional to the density of invasion. We constructed microcosms with mixtures of native and invasive leaf litter, and measured microbial community structure (phospholipid fatty acids) and function (litter decomposition). Decomposition was linearly related to the degree of invasion (R 2?=?0.945), but the ratio of bacteria to fungi exhibited a strongly non-linear, threshold response (R 2?=?0.513). These results indicate that impacts of Japanese barberry invasion are not always proportional to invasion density. This finding has implications for the study of biological invasions as well as practical implications for the management of exotic invasive species. 相似文献
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90.
Selectivity of Ferric Enterobactin Binding and Cooperativity of Transport in Gram-Negative Bacteria 总被引:1,自引:5,他引:1 下载免费PDF全文
Padmamalini Thulasiraman Salete M. C. Newton Jide Xu Kenneth N. Raymond Christine Mai Angela Hall Marjorie A. Montague Phillip E. Klebba 《Journal of bacteriology》1998,180(24):6689-6696
The ligand-gated outer membrane porin FepA serves Escherichia coli as the receptor for the siderophore ferric enterobactin. We characterized the ability of seven analogs of enterobactin to supply iron via FepA by quantitatively measuring the binding and transport of their 59Fe complexes. The experiments refuted the idea that chirality of the iron complex affects its recognition by FepA and demonstrated the necessity of an unsubstituted catecholate coordination center for binding to the outer membrane protein. Among the compounds we tested, only ferric enantioenterobactin, the synthetic, left-handed isomer of natural enterobactin, and ferric TRENCAM, which substitutes a tertiary amine for the macrocyclic lactone ring of ferric enterobactin but maintains an unsubstituted catecholate iron complex, were recognized by FepA (Kd ≈ 20 nM). Ferric complexes of other analogs (TRENCAM-3,2-HOPO; TREN-Me-3,2-HOPO; MeMEEtTAM; MeME-Me-3,2-HOPO; K3MECAMS; agrobactin A) with alterations to the chelating groups and different net charge on the iron center neither adsorbed to nor transported through FepA. We also compared the binding and uptake of ferric enterobactin by homologs of FepA from Bordetella bronchisepticus, Pseudomonas aeruginosa, and Salmonella typhimurium in the native organisms and as plasmid-mediated clones expressed in E. coli. All the transport proteins bound ferric enterobactin with high affinity (Kd ≤ 100 nM) and transported it at comparable rates (≥50 pmol/min/109 cells) in their own particular membrane environments. However, the FepA and IroN proteins of S. typhimurium failed to efficiently function in E. coli. For E. coli, S. typhimurium, and P. aeruginosa, the rate of ferric enterobactin uptake was a sigmoidal function of its concentration, indicating a cooperative transport reaction involving multiple interacting binding sites on FepA. 相似文献